1. Field of the Invention
The invention relates to modulation of gene expression. In particular, the invention relates to modulation of gene expression through an antisense approach.
2. Summary of the Related Art
Regulation of gene expression is a complex process, and many aspects of this process remain to be understood. Aberrant gene expression appears to be responsible for a wide variety of inherited genetic disorders, and has also been implicated in numerous disease states including pathological conditions stemming from tumorigenic growth. A great deal of cancer related research pertains to the elucidation of the roles and interaction of tumor suppressor genes and oncogenes. For instance, tumor growth and malignancy are known to result from the combination of 1) activation of proto-oncogenes that promote cell growth with 2) inactivation of tumor suppressor genes that inhibit cell growth through cell growth arrest and induction of programmed cell death or apoptosis.
Several tumor suppressors have been identified. For instance, the identification and isolation of the WT1 gene is taught in Marshall et al., Cell 64, 313-326 (1991). Coopers et al., Cancer Invest. 12(1), 57-65 (1994) discloses that the WT1 gene product is a protein with four zinc fingers suspected to be a transcription factor. Further, Anderson and Spandidos Onco-Suppresso (1990) disclose the NF1 gene, another tumor suppressor, involved in the development of neurofibromatosis functioning as a GTPase-activating protein for the GTP-binding protein p21ras. In addition, Sager et al. Science 246, 1406-1412 (1989) discloses several genes involved in the development of colon cancer, namely DCC, MCC and APC (FAP), suggesting that their products might also perform tumor suppressor functions.
To date however, the best characterized tumor suppressors are the RB and the p53 gene products (See, for example, “The p53 Pathway”, Prives et al J. Pathol. 187, 112-126 (1999)). Levine Bioessays 12(2), 60-66 (1990) teaches RB gene inactivation in retinoblastoma. Notably, Levine et al. Nature 351, 453-456 (1991), Weinberg et al. Neur. 11, 191-196 (1991), and Williams et al. Nature Genet. 7, 480-484 (1994), teach RB gene inactivation in many other tumor types including breast tumors, bladder carcinoma, and lung tumors.
Levine et al. Nature 351, 453-456 (1991) teaches the p53 tumor suppressor gene's ability to encode a phosphoprotein suspected to play a pivotal role in fundamental biological processes in cell proliferation and differentiation. Lane Br. Med. Bull. 50, (3)582-599 (1994) also teaches the p53 gene involvement in various types of tumors. In addition, Lowe et al. Cell 74, 957-967 (1993) discloses that p53 is required to trigger apoptosis in response to chemotherapy and that p53 activation is an important factor in mediating the cytotoxic effects of many cancer treatments, including chemotherapy and radiation. See also Lowe et al. Science 266, 807-810 (1994); Kastan et al. Cancer Res. 51, 6304-6311 (1991); and Fritsche et al. Oncogene 8, 307-318 (1993).
Further elucidation of the role of both RB and p53 regulation has led to the discovery that the mouse double-minute, or MDM2 oncogene is a negative regulator of wild-type p53 (Fakharzadeh et al., EMBO J. 10:1565-1569 (1991); Piette et al., Oncogene 15:1001-1010 (1997)). The human cDNA sequence (SEQ ID NO: 1) is disclosed in Volgelstein and Kinzler, U.S. Pat. No. 5,411860 and the mouse cDNA sequence (SEQ ID NO: 12) can be found in GenBank, Accession No. U40145. Cahill-Snyder et al., Somatic Cell. Mol. Genet. 13:235-244 (1987) teach the identification of this oncogene because of its overexpression in a spontaneously transformed tumor cell line.
The MDM2-p53 autoregulatory feedback loop regulates the intracellular p53 function: the MDM2 gene is a target for direct transcriptional activation p53 and MDM2 protein is a negative regulator of p53. In addtition, MDM2 protein interacts with other cellular proteins that are involved in cell cycle regulation, including pRB, E2F1/DP1, p300 and p19ARF.
Overexpression of MDM2 is demonstrated in a variety of human tumors and may be due to one or more of three mechanisms: 1) gene amplification; 2) increased transcription; and/or 3) enhanced translation. Several studies have shown that overexpression of MDM2 is associated with poor prognosis in many human malignancies. Therefore, MDM2 plays a crucial role in cell cycle control and tumor transformation and growth.
The significance of MDM2 in cell regulatory functions has been extended to other interactions as well. Marechal et al., Mol. Cell. Biol. 14:7417-7429 (1994) teaches the binding of the MDM2 protein to the ribosomal protein L5-5S RNA complex while Elenbaas et al., Mol. Med. 2:(4)439-451 (1996) teaches MDM2 interaction with specific RNA structures.
Gastrointestinal cancers remain a major public health problem both in the USA and worldwide. In the United States, colorectal cancer is the second most common cancer in women and the third most common cancer in men. Although there has been considerable progress in research on the etiology, prevention, and experimental therapy of gastrointestinal cancers, no fully effective approaches are available currently for the treatment and prevention of this disease. MDM2 overexpression has been observed in human colorectal cancer.
MDM2 has been suggested as a novel target for cancer therapy, especially the p53-mdm2 interaction. The rationale for this at least includes the following: 1) MDM2 amplification and overexpression occur in many types of human cancers and the MDM2 levels correlate with poor prognosis in some cancers; 2) p53, which is negatively regulated by MDM2, plays a major role in tumor growth; 3) p53-mediated growth arrest and/or apoptosis have been suggested to be major mechanisms for currently used cancer therapy such as DNA damaging chemotherapeutics and radiation therapy; 4) loss of p53 function and/or overexpression of MDM2 is believed to correlate with tumor resistance to conventional therapy; and 5) MDM2 has displayed both p53-dependent and p53-independent activities in connection with its tumorigenic property. In the past few years, several strategies have been used to test the hypothesis that, by disrupting p53-MDM2 interaction, the negative regulation of p53 by MDM2 is diminished and the cellular functional p53 level will be increased, particularly following DNA damaging treatment, resulting in tumor growth arrest and/or apoptosis that leads to better therapeutic response. These approaches include the use of polypeptide, antibody, and antisense oligonucleotides.
Recently, we have successfully identified an anti-MDM2 antisense PS-oligo that effectively inhibits MDM2 expression in tumor cells containing MDM2 gene amplifications (Chen L. et al., Proc Natl Acad Sci USA, 95: 195-200, 1998). Effective anti-human-MDM2 antisense PS-oligos were initially screened in two cell lines, JAR (choriocarcinoma) and SJSA (osteosarcoma), that contain wild type p53, amplified MDM2 genes, and overexpression of MDM2 oncoprotein. Of nine PS-oligonucleotides screened, Oligo AS5 (5′GATCACTCCCACCTTCAAGG-3′; SEQ ID NO:28), which can hybridize to a position ˜360 bp downstream of the translation start codon, was found to reproducibly decrease MDM2 protein levels in both cell lines by 3-5 fold at concentrations of 100-400 nM in the presence of Lipofectin. The mismatched control Oligo M4 (5′-GATGACTCACACCATCATGG-3′; SEQ ID NO:5) had no effect on MDM2 expression. Oligo AS5 was also shown to induce RNase H cleavage of the target MDM2 mRNA, resulting in truncation and degradation of the target. Further studies demonstrated that, following AS5 treatment, the p53 protein level was elevated and its activity was increased. A dose-dependent induction of p21 expression by AS5 was observed up to 6.6 fold at the optimal concentration of 200 nM, suggesting that p53 transcriptional activity be increased following inhibition of MDM2 expression. JAR cells treated with AS5 showed a significant increase in the levels of apoptosis. AS5 did not cause visible apoptosis in the H1299 cells that lack p53. These results suggested that apoptosis induced by AS5 is due to activation of p53 following MDM2 inhibition by the oligonucleotide.
In general, human cancer cell lines or tumor tissues with MDM2 gene amplifications or overexpression often have wild-type p53, presumably inactivated by MDM2. Several studies have now shown that overexpression of MDM2 is associated with poor prognosis in human malignancies, including osteosarcoma, soft tissue sarcoma, breast cancer, ovarian cancer, cervical cancer, oral squamous cell carcinoma, brain tumor, esophageal cancer, colorectal carcinoma, bladder cancer, urithelial carcinoma, leukemia, and large B cell lymphoma. These studies suggest that overexpression of MDM2 is associated with inactivation of wild-type p53, the inhibition of MDM2 expression in these tumors may lead to re-activation of p53 and induction of cell growth arrest of apoptosis of human tumors. It has been demonstrated that many cancer therapeutic agents exert their cytotoxic effects through activation of wild-type p53, and the restoration of wild-type p53 can increase the sensitivity of tumors to DNA-damaging agents. Restoration of wild-type p53 may also overcome the drug resistance of human cancers associated with dysfunction of p53. However, the activation of p53 by DNA damage such as cancer chemotherapy and radiation treatment may be limited in cancers with MDM2 expression, especially those with MDM2 overexpression. Therefore, the inactivation of the MDM2 negative feed-back loop may increase the magnitude of p53 activation following DNA damage, thus enhancing the therapeutic effectiveness of DNA damaging drugs.
In addition to its interaction with p53, MDM2 has also been shown to bind to and interact with other cellular proteins such as the pRB, E2 Fl, p300, ARF, p73, Numb and ribosomal protein, and RNA. Also, MDM2 has been shown to regulate the MyoD transcription factor. The biological consequences of these activities are not fully understood, but may be associated with transforming properties of MDM2 that may be p53-independent.
p53-Independent activity of MDM2 has been suggested by several reports and reviews. MDM2 gene products include several forms of polypeptide, representing alternatively spliced MDM2 variants. Various alternatively spliced MDM2 polypeptides have been found in several human tumors. Of the five forms of MDM2 analogs, only one retains p53 binding capability. However, cDNAs coding for all five forms of alternatively spliced MDM2 could independently transform NIH3T3 cells, indicating that these MDM2 transcripts have the p53-independent transforming ability. The effects of MDM2 overexpression on mammary tumorigenicity are seen in p53-null mice, indicating that MDM2 can cause transformation and tumor formation via a p53-independent mechanism. More recently, overexpression of MDM2 is shown to be associated with resistance to the antiproliferative effects of transforming growth factor β (TGF-β), which is p53-independent.
One of the advantages of the use antisense oligonucleotides or MDM2 specific antibody is that these agents may exert their effects in all MDM2 expressing tumors regardless of p53 status. This is very important since the p53-independent activity of MDM2 may play a role in MDM2 tumorigenicity. Inhibition of MDM2 expression will ultimately prevent the interaction of MDM2 and other cellular protein. For example, the recent development of certain antisense anti-MDM2 oligonucleotide (Chen L. et al., Proc Natl Acad Sci USA, 95: 195-200, 1998) has been shown to increase E2F-1 levels following microinjection (Blattner C. et al., Mol. Cell. Biol. 19: 3704-3713, 1999).
One potential drawback is that these agents may have similar effects on normal host tissues, resulting in activation of endogenous p53 in normal tissues. The tolerance of increased p53 levels in normal tissues will be the key for the success of approaches aiming at elimination of MDM2 from the cells. The activation of p53 in normal tissues following DNA damaging treatment and resultant cell growth arrest and apoptosis are believed to be associated with side toxicities of conventional therapy. A recent study demonstrates that inhibition of p53 function can in fact prevent host toxicity associated with DNA damage treatment.
From the available literature, it is clear that efforts should be directed to identify modulators and potentiators of tumor suppressor genes expression as a possible therapeutic approach to tumorigenesis. The identification of regulatory proteins acting on tumor suppressors could potentially lead to the development of therapeutic approaches to tumorigenesis by the activation of tumor suppressor functions. Thus, there is a need for the identification of tumor suppressor regulators and of methods to activate tumor suppressors in the context of chemotherapy. In this context, there is a need to elucidate the mechanism(s) involved in the development of resistance to chemotherapy in tumor cells. There is therefore, a need to develop better tools to carry out such investigations. Ideally, such tools should take the form of improved antisense oligonucleotides that inhibit MDM2. Kondo et al., Oncogene 10:(10)2001-2006 (1995) has disclosed that antisense oligonucleotide phosphodiesters directed against MDM2 increase the susceptibility of tumor cells to cisplatin-induced apoptosis. Kondo et al. have recently disclosed that MDM2 gene induced the expression of the multidrug resistance gene (mdr1) and that of its product P-glycoprotein (P-gp) conferring resistance to the apoptopic cell death induced by DNA-damage inducing drugs. Kondo et al., Br. J. Cancer 74:(8)1263-1268 (1996) teach the antisense inhibition of the MDM2 gene to inhibit expression of p-gp in MDM2 expressing glioblastoma cells further suggesting that the MDM2 gene may play an important role in the development of MDR phenotype in human tumors. Unfortunately the oligonucleotides disclosed are phosphodiester oligonucleotides and thus not suitable as investigative tools for the purposes discussed herein, and as potential therapeutics for the treatment of neoplastic diseases. Therefore, there remains a need for improved antisense oligonucleotides. Such improved antisense oligonucleotides should preferably also represent potential therapeutics for the treatment of neoplastic disease.